DNase I and Hydroxyl Radical Characterization of Chromatin Complexes

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

The native chromatin complex within most eukaryotic nuclei is very difficult to study by biochemical means, so researchers have developed methods for studying smaller portions of the complex. This unit details the use of DNase I and hydroxyl radicals to characterize histone?DNA interactions within such portions of the complex. DNase I digestion can be used to determine what regions of a DNA segment are intimately associated with the core histone proteins and what regions are more like naked DNA (i.e., linker DNA within the nucleosomal repeat). The finer deatils of histone?DNA interactions and DNA structure within these complexes is best characterized by digestion with the hydroxyl radical. Both reagents may be used to assess the degree and homogeneity of rotational and translational positioning within isolated chromatin complexes.

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Basic Protocol 1: Analysis of Chromatin Complexes by DNase I Cleavage
Alternate Protocol 1: Footprinting with Hydroxyl Radicals
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Analysis of Chromatin Complexes by DNase I Cleavage

Materials

Nucleosome preparation, radiolabeled (see )
recipeNucleosome buffer (see recipe )
20 mM MgCl₂
2000 U/ml (1 mg/ml) DNase I: purchase 10,000 U lyophilized with glycine (Worthington) and reconstitute with 5 ml H₂ O (store in aliquots at −70°C)
10× stop solution: 50 mM EDTA /0.2% (w/v) SDS (store up to 6 months at room temperature)
3 M and 0.3 M sodium acetate ( appendix 22 )
95% and 70% ethanol (−20°C)
0.3 M sodium acetate in TE buffer, pH 8.0
10 mg/ml Pronase or 10 mg/ml protease K (store up to 1 year at −20°C)
2% (w/v) SDS
0.1% (w/v) SDS in TE buffer, pH 8.0
25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol made with buffered phenol (unit 2.1 )
25 mM EDTA /50% (v/v) glycerol
0.7% preparative agarose gel (unit 2.5 ) in recipe1× Tris⋅borate buffer, pH 8.3 (see recipe )
Running buffer for nucleoprotein gel electrophoresis: recipe1× Tris borate buffer, pH 8.3 (see recipe )
TE buffer, pH 8.0
100% formamide containing 1 mM EDTA and 0.005% tracking dyes

Transfer micropipets
90°C water bath

Additional reagents and equipment for phenol extraction and ethanol precipitation of DNA (unit 2.1 ), agarose gel electrophoresis (unit 2.5 ), autoradiography and phosphor imaging ( appendix 3A ), and sequencing gel elecrophoresis (unit 7.6 )

NOTE: Proper sample preparation is critical for both the DNase I and hydroxyl radical cleavage procedures. See for guidelines before proceeding with the experiments.

Alternate Protocol 1: Footprinting with Hydroxyl Radicals

Glycerol
1 M Tris⋅Cl, pH 8.0 ( appendix 22 )
recipeIron/EDTA working solution (see recipe ), freshly prepared
10 mM sodium ascorbate [prepare fresh from 1 M stock stored at −70°C (stable >1 year; e.g., Aldrich)]
0.12% H₂ O₂ , prepared fresh by 1:250 dilution from 30% H₂ O₂ (stock solution stable up to several years at 4°C if kept free of metal contamination)

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Figures

Figure 21.4.1 Flow chart for DNase I and hydroxyl radical cleavage analysis of chromatin complexes.

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Literature Cited

Literature Cited
	Hansen, J.C. 1997. The core histone amino termini: Combinatorial interaction domains that link chromatin structure with function. Chemtracts 10:737‐750.
	Hayes, J.J., Tullius, T.D., and Wolffe, A.P. 1990. The structure of DNA in a nucleosome. Proc. Natl. Acad. Sci. U.S.A. 87:7405‐7409.
	Hayes, J.J., Bashkin, J., Tullius, T.D., and Wolffe, A.P. 1991. The histone core exerts a dominant constraint on the DNA in a nucleosome. Biochemistry 30:8434‐8440.
	Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F., and Richmond, T.J. 1997. Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature 389:251‐260.
	Schwartz, P.M., Felthauser, A., Fletcher, T.M., and Hansen, J.C. 1996. Reversible oligonucleosome self‐association: Dependence on divalent cations and core histone tail domains. Biochemistry 35:4009‐4015.
	Suck, D. and Oefner, C. 1986. Structure of DNase I at 2.0 Å resolution suggests a mechanism for binding to and cutting DNA. Nature 321:620‐625.
	Tullius, T.D. 1987. Chemical snapshots of DNA: Using the hydroxyl radical to study the structure of DNA and DNA‐protein complexes. Trends Biochem. Sci. 12:297‐300.
	Tullius, T.D. and Dombroski, B.A. 1985. Iron(II) EDTA used to measure the helical twist along any DNA molecule. Science 230:679‐681.
	Tullius, T.D., Dombroski, B.A., Churchill, M.E., and Kam, L. 1987. Hydroxyl radical footprinting: A high‐resolution method for mapping protein‐DNA contacts. Methods Enzymol. 155:537‐558.
	van Holde, K.E. 1989. Chromatin. Springer‐Verlag, New York.
	Wolffe, A.P. 1995. Chromatin: Structure and Function. Academic Press, London.
	Wolffe, A.P. and Hayes, J.J. 1993. Transcription factor interactions with model nucleosome templates. Mol. Genet. 2:314‐330.